Inhibition of monoacylglycerol lipase mediates a cannabinoid 1-receptor dependent delay of kindling progression in mice
Introduction
Anandamide and 2-arachidonylglycerol (2-AG), the two most studied endocannabinoids in the brain, are small lipid molecules, which retrogradely traverse the synapse and act presynaptically on metabotropic cannabinoid type 1 receptors (CB1Rs) (Kreitzer and Regehr, 2001, Maejima et al., 2001, Wilson and Nicoll, 2001). The activation of CB1Rs inhibit adenylyl cyclase and voltage-gated Ca2+ channels, resulting in a decreased release of neurotransmitters, thus modulating neuronal excitation and inhibition (Bidaut-Russell et al., 1990, Sugiura et al., 1997, Lauckner et al., 2005). As a consequence, the activation of the endocannabinoid system may be a safeguard against hyperexcitability, acute seizures and excitotoxity (Ameri and Simmet, 2000, Marsicano et al., 2003, Monory et al., 2006), raising the idea to slow down or even to prevent the development or progression of epilepsy. This is of particular interest, when considering that during excessive excitation endocannabinoid signaling has stronger effects on cortical principal neurons than on GABAergic interneurons (von Rüden et al., 2015). Therefore, the endocannabinoid system is discussed as a potential target for the prevention of epilepsy (Hofmann and Frazier, 2013). The process of epilepsy development (= epileptogenesis) refers to molecular and cellular alterations, which transform a physiological neuronal network into an epileptic state, with an increased risk of recurrent spontaneous seizures (Goldberg and Coulter, 2013). The detailed etiology of epileptogenesis and if the endocannabinoid system may be involved in epileptogenesis remains incompletely understood.
There are only few experimental studies in rodents which indicate anti-epileptogenic properties of direct CB1R activation (Bhaskaran and Smith, 2010, Wendt et al., 2011). Recently, we evaluated the impact of the CB1R agonist WIN55.212-2 as well as of the indirect agonist URB597 on kindling progression (Wendt et al., 2011). WIN55.212-2 delayed kindling acquisition whereas URB597, which inhibits the catabolic enzyme fatty acid amide hydrolase and thereby reduces the degradation of anandamide, had no effect on the generation of a hyperexcitable neuronal network. In contrast to anandamide, 2-AG acts as a full agonist on CB1Rs and exhibits higher CB1R efficacy (Sugiura et al., 1999, Sugiura et al., 2000, Sugiura et al., 2006). A molecular and morphological study of mossy cell-granule cell synapses revealed that 2-AG mediates retrograde signaling at these synapses and may reduce the excitability of granule cells and prevent seizures (Uchigashima et al., 2011).
Although these experimental data are only limited, they strongly suggest a potential therapeutic role for the endocannabinoid 2-AG in epilepsy. However, there is, to our knowledge, no information about the impact of 2-AG on seizure thresholds or on the progression of seizure development in epilepsy. Considering this information, it is of particular interest to evaluate whether increased 2-AG levels affect these parameters in a chronic epilepsy model with excellent predictive validity for temporal lobe epilepsy.
The aim of this study is to evaluate if pharmacological inhibition of monoacylglycerol lipase (MAGL) by JZL184 has an impact on the progression of epileptic seizures in the kindling mouse model of temporal lobe epilepsy and to demonstrate that JZL184-mediated effects are CB1R dependent. For this purpose, the indirect CB1R agonist JZL184 was used. JZL184 potently and selectively inhibits the 2-AG degrading enzyme MAGL and this results in increased 2-AG levels up-to an 8-fold (Long et al., 2009). This strategy offers a clear benefit compared with direct CB1R agonism namely event-specific effects, which are based on inhibition of the endocannabinoid degrading enzyme at CB1Rs localized on excessively activated glutamatergic synapses.
Section snippets
Animals
Animal experiments have been performed in accordance with the EU directive 2010/63/EU and with the German Animal Welfare act. They were approved by the responsible government (license numbers 55.2-1-54-2532-93-11 and 55.2-1-54-2532-173-11). Male mice with a body weight of 21–15 g were used in all experiments and maintained in standard conditions with food and water ad libitum (24–25 °C; humidity 50–60%; lights on from 7 am to 7 pm). NMRI mice were acquired from Harlan Netherlands (Horst,
JZL184 administration increased the afterdischarge threshold at the beginning of the kindling process
Afterdischarge thresholds were determined at the beginning of the kindling process (under JZL184 treatment) and again at the end of kindling after a washout time of 6 days. Treatment with JZL184 at the beginning of kindling significantly increased the afterdischarge threshold by 233% (p = 0.0325; Fig. 2A) in NMRI mice. Afterdischarge threshold termination at the end of kindling without JZL184 administration revealed no longtime effect between the two groups (p = 0.8939; Fig. 2A). As expected,
Elevated 2-AG levels delay the development of generalized epileptic seizures
In the present study, we demonstrate that JZL184 treatment increases seizure thresholds in naïve NMRI mice and delays the development of generalized epileptic seizures in the kindling model. JZL184 treatment strongly increases the concentration of 2-AG in the brain by a selective and effective block of the catabolizing enzyme MAGL (Long et al., 2009, Schlosburg et al., 2010, Wiskerke et al., 2012). Although we did not measure brain 2-AG levels in our experiment, the observed effects confirm
Conclusion
The aim of this study has been to evaluate whether pharmacological inhibition of MAGL by JZL184 affects the development and progression of epileptic seizures in the kindling mouse model of temporal lobe epilepsy and to prove that JZL184-mediated effects are CB1R dependent. Our data demonstrate that indirect CB1R agonism can interfere with the development of generalized epileptic seizures in the kindling model but has no anticonvulsive effects. Furthermore, we confirmed that the effects of
Conflict of interest
All authors disclose any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within 3 years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work.
Acknowledgments
The authors are grateful to Beat Lutz for providing the genetically modified mice and to Elite Netzwerk Bayern e.V. for financial support of Eva-Lotta von Rüden.
The authors acknowledge the excellent technical support of Olga Cabezas, Marion Fisch, Barbara Kohler, Claudia Siegl, Angela Vicidomini, Andrea Reßle and the MPG breeding facilities in conducting the animal experiments and data collection.
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